JPH09252788A - Human mucin (muc8) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new cDNA for production, etc., of diagnostic or vaccine for detection of antibody against mucin as a tumor marker coding human mucin MUC8 corresponding to a nucleotide sequence of pCos nucleotide.

SOLUTION: This new cDNA codes human mucin MUC corresponding to nucleotide sequence of pCos nucleotide 3049 to 4328 represented by the formula and is useful as a diagnostic agent for detecting an antibody against mucin as a tumor marker and vaccine for treating diseases due to immunity resistant stimulation. cDNA coding the human mucin MVC8 is obtained by extracting mRNA from a colon carcinoma cell system T84 according to ordinary method, preparing human cDNA gene expression library, screening the library by using an antibody against human mucin MVC8, selecting positive plague and cloning the plague and recovering the DNA.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the isolation and characterization of amino acid and nucleotide sequences of novel members of the mucin gene family. In particular, the invention relates to the manufacture of reagents for vaccination for the purpose of diagnosing patients and treating certain diseases by stimulating immune resistance.

[0002]

The epithelium of the respiratory, genital and gastrointestinal tracts of higher animals is covered by a protective secretion called mucus. This mucus gel is 95% water and approximately 5% mucin (Neutra & Forstner, 1987). Mucin is a glycoprotein with two specific characteristics. That is, firstly at least 50% of their molecular weight is formed from oligosaccharides linked by O-glycosidic bonds to residues of threonine and serine of the protein backbone, and secondly in this highly glycosylated region. Is to be composed of repeating sequence units. Mucins can be divided into two groups. One is a secretory mucin that exists as an oligomer linked by intermolecular disulfide bridges, and the other is a membrane-localized mucin that is anchored in the plasma membrane by a hydrophobic region (Gum, 1992; Metzgar Et 1
993; Strous & Dekker, 1992).

Since 1987, the DNA sequences of seven members of the mucin family have been isolated and characterized from human gene libraries. Comparing the sequences of different mucins, no significant homology was observed in either their nucleotide composition or their amino acid composition. However, a feature common to all mucins is the central region, which has O-glycans and is composed of a continuous repeating nucleotide sequence and amino acid motif.

Changes in mucin expression and post-translational modification are associated with various diseases such as cancer, cystic fibrosis, ulcerative colitis and localized ileitis (Crohn's disease), which has become the subject of clinical studies. ing. Antibodies that recognize mucin epitopes (CA19-9, CA125 and M2
6) is used as a marker in diagnosis (Ma
gnani et al., 1984; Metzgar et al., 1984; Linsley et al., 198.
8).

Many researchers have attempted to gain new information on mucin polypeptide and nucleic acid sequences. Despite advances in protein chemistry, it is often impossible to purify high molecular weight mucin glycoproteins,
Also, the use of recombinant DNA technology in the case of highly repetitive nucleotide sequences is problematic, thus placing great demands on the discovery of yet another mucin that could lead to advances in patient diagnosis and treatment. There is.

[0006]

The object of the present invention is therefore to discover further mucins in order to increase the overall diagnostic value of mucins and thereby achieve an important contribution to tumor diagnosis. It was based on.

[0007]

This object is achieved by the provision of the embodiments described in the claims, in particular in the production of the novel mucins and mucin genes.

The invention presented here allows the use of a nucleotide sequence encoding a novel human mucin. The nucleotide sequences are present as genomic DNA and cDNA inserted in a circular plasmid vector. These constructs containing mucin sequences were used for stable transformation of bacterial host organisms (E. coli), allowing these organisms to infinitely amplify mucin sequences.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION By expressing a coding sequence in a prokaryotic expression system, a recombinant polypeptide can be produced and purified in large quantities without contaminating other human glycoproteins. Bacterial hosts can be cultivated on a large scale without difficulty. Since this polypeptide is synthesized as a fusion protein conjugated with maltose binding protein (MBP), it can be isolated from cell lysates by production affinity chromatography using Mab B1 or maltose. Expression of the polypeptide can be promoted in a specific manner by induction of the bacterial promoter with IPTG.

This particular polypeptide allows the establishment of an ELISA for the screening of patient sera and can be used for the production of antibodies.

A sandwich ELISA system, which allows the detection of other antigenic determinants on the native human MUC8 glycoprotein by binding other specific antibodies to the solid phase, provides the antibody with alkaline phosphatase or horseradish peroxidase. It can be developed by coupling.

In this way, the novel invention can be utilized for the development of a diagnostic kit that can be routinely used clinically.

Since the MUC8 gene was revealed to exist on human chromosome 7 by Southern blot analysis, the labeled (radiolabeled or non-radiolabeled with biotin) DNA probe was used for the adjacent gene or the corresponding It is suitable for use as a genetic marker for the identification of mutations (deletions, translocations and insertions). MUC8
Genes are highly polymorphic, as revealed by hybridization with DNA probes from different patients, so using MUC8 DNA as a probe in fingerprint analysis to confirm family relationships. You can

Yet another embodiment is the MU in a specific manner.
Antisense R that allows blocking of C8 mRNA translation
Use of NA or antisense oligonucleotides. This indicates that expression of mucin by tumor cells leads to an increased metastatic rate in animal models (Bresalier et al., 199).
From 1), it may be possible to improve the prognosis of the tumor. Vaccination experiments also promise other potential applications. In the case of the MUC1 peptide, the mucin epitope has been shown to be recognized by cytotoxic T cells in a manner that is not MHC-restricted and thus capable of eliciting a tumor-specific immune response (Hilkins et al., 1992). ; B
arnd et al., 1989; Ioannides et al., 1993).

The expression pattern of B1 antigen in humans was determined by immunohistochemical examination of cryostat sections of various benign and malignant tissues. From this data, M
A site that can be used in a diagnostic system for detecting UC8 mucin can be estimated.

The nucleotide sequence encoding human MUC8 mucin has been isolated within the scope of the invention described herein. Portions of repetitive nucleotide sequences have been expressed with the purpose of allowing the isolation and study of relatively large amounts of polypeptides using their biological and immunological properties.

The DNA used for this purpose was IZAP c prepared from mRNA of the human colon cancer cell line T84.
It was isolated by immunoscreening the DNA gene library. This clone was designated as pbl and contains 798 bp. The cDNA contains a 282 bp 3'-untranslated region, which is composed of Alu repeats, contains a polyadenylation signal, and is poly (A).
It ends in a chain. The coding sequence for 517 nucleotides is 53
It is composed of bp repeat units and terminates at the opal stop codon. Ec of this cDNA containing the code repeat region
The oRI / HindIII fragment was used as a probe in Northern blot analysis. In human tumor cell lines, 4.8 kb, 1.9 kb and 1.35 kb
3 of the three mRNA species hybridize, among which 4.
The 8 kb band produced the strongest signal. Eco
EcoR I by Southern blot analysis performed on human genomic DNA samples digested with R I and Msp I
It was possible to observe restriction fragment length polymorphisms when the / HindIII fragment was used as a probe, which was most apparent in the case of the Msp I-digested sample. To obtain further sequence information, EcoR
Phage I using the I / Hind III fragment
The human kidney cDNA gene library in MAXI and the genomic gene library in cosmid vector pWE-15 were screened. A 854 bp long cDNA fragment was isolated from the IMAXI gene library. This clone, pB1.2 is 576 bp
Contains the untranslated region, followed by a 278 bp coding sequence after the codon believed to be the initiation codon. When the genomic gene library was screened, a positive clone, pWE-B1 was isolated and D was inserted into it.
NA was digested with various restriction enzymes and analyzed by Southern blot. A BglII / BamHI fragment of 5.9 kb in length hybridized with either B1 cDNA, which was further characterized by pBSK (+).
Subcloned into. The resulting clone pB1-C
The os genomic DNA was completely sequenced and the nucleotide sequence was compared to two cDNA clones. Homology comparison reveals that the pB1-Cos clone contains an exon of 1482 bp and its 5'region is identical to that of the pB1.2 clone, while the 3'region corresponds to that of the pB1 clone. Was done. The central region of the exon, which is not represented by the cDNA nucleotide sequence, is composed of additional repeating units, as shown in the partial digestion analysis using the restriction enzyme Ear I. pB
All 1-Cos clones are 53 bp long Ear
Contains 19 II subfragments. By Southern blot using DNA from somatic cell hybrids,
The locus of the UC8 gene could be assigned to chromosome 7.

The DNA fragment encoding MUC8 mucin could be inserted into a eukaryotic expression vector and used for transformation (stable or transient) into mammalian cells, especially human cells. New generation vectors usually provide enhancer and promoter sequences and allow selection in both prokaryotic and eukaryotic systems, thus ensuring easy manipulation by expression of MUC8 in eukaryotic cells. This way, native MUC
Co- or post-translationally modified polypeptides, which should correspond to 8 mucins, can be isolated and analyzed. This construct can be stably introduced into human B lymphocytes immortalized with Epstein-Barr virus in order to be able to provide MUC8 peptide fragments with MHC classes I and II. Expression of tumor-associated epitopes can be increased even further by incubating transfected cells with inhibitors of post-translational O-glycosylation (Bu et al., 1993). MUC
In the case of 1 mucin, it has repeatedly been demonstrated that due to the mucin repetitive structure, cytotoxic T cells recognize the mucin epitope in a manner not dependent on MHC restriction. The idea here is to reintroduce a transfectant expressing high amounts of MUC8 into a patient in order to elicit a specific tumor associated response by antibody or CTL recognition.

Another aspect is the detection of tumor or inflammation in a patient by detecting the mucin antigen in tissue or serum. Simple histology such as immunohistochemical staining of tissue sections, ELISA with patient sera, or Western blot using the hybridoma cell line B1 which allows virtually unlimited isolation of antibodies from culture supernatants. Method becomes possible. Hybridoma is Balb /
c mice were immunized with a high molecular weight protein fraction from Mz-Sto-1 cell extracts and then isolated by fusing the spleen cells of immunized mice with immortalized myeloma cells (Herbst
hofer, 1990). The gastric adenocarcinoma cell line Mz-Sto-1 was established from signet ring cell carcinoma patients (Dippold, 1987). M of sera from patients with pancreatic cancer and having elevated CA19-9 levels compared to sera of colon cancer patients and healthy donors
Elevated levels of UC8 were revealed by Western blot analysis. This provides the first indicator for establishing MUC8 as a tumor marker in patient sera.

N-after incubation with IPTG
PB1cD in vector pMAL-c enabling expression of a fusion protein having a terminal maltose binding protein part
As a result of the expression of NA, it is recognized by the B1 antibody,
Polypeptides believed to contain antigenic determinants are therefore available. Computer analysis shows that the hexamer QDREEE (one letter abbreviation) is most likely the epitope. Consists of 53 amino acid residues, 159
Each repeating unit encoded by a nucleotide base pair contains this peptide. The latter is therefore MUC8
It can be used in immunological methods to specifically block the recognition of antigens.

MUC8 polypeptides which can be isolated following expression in prokaryotic or eukaryotic cells can be used directly as protective factors for various diseases (eg ulcers) and also for the generation of MUC8 specific antibodies. it can. If the polypeptides are larger than 10 kD, they can be used as such as immunogens, but otherwise they need to be coupled with a carrier protein to ensure effective immunity. Peptide to vertebrates (mouse, rat,
MUC8-specific antibodies can be isolated from animals as polyclonal antisera after injection into rabbits, goats or sheep). For the production of the monoclonal antibody, the method of Milstein & Kohler effectively used for the production of Mab B1 must be adopted. Antibodies can be purified from culture supernatants of niblyoma cell lines using an appropriate protocol optimized for each subclass (Harlow,
E. & Lane, D. “Antibodies: A Laboratory Manual”
(1988), Cold Spring Harbor, New York). Polypeptides and antibodies can be modified by covalently or non-covalently conjugating to substances that allow detection to be performed. These substances can be radionuclides, enzymes, cofactors, magnetic particles, fluorescent agents or chemiluminescent agents. These modified antibodies or polypeptides can be conjugated to biological materials such as tissue biopsy specimens, blood,
It can be used in various immunological methods for detecting MUC8 in urine, serum and other body fluids. Tissue samples are routinely examined as paraffin sections or cryostat sections on slides with labeled MUC8 antibody or unmodified antibody whose binding must be detected with a second labeled antibody. Proteins from body fluids can be tested in a simple manner by ELISA system and Western blot analysis. Numerous competitive and non-competitive immunological test systems are known for coupling an antigen or antibody to a solid phase (Harlow & Lane, 1988).

For use in the treatment of cancer, the expression of MUC8 in order to specifically injure or label tumor tissue by coupling humanized MUC8-specific antibodies to toxins or radionuclides as much as possible. It can be administered to patients with elevated levels.

The abbreviations used herein have the following meanings. bp base pair BSA bovine serum albumin h time kD kilodalton MBP maltose binding protein RT room temperature

[0024]

The following examples illustrate the invention.

Example 1 A) Screening of phage gene library A human cDNA gene expression library was phage Uni
-ZAP XR (Stratagene) prepared from mRNA of middle colon cancer cell line T84. Phages were plated in soft agarose on a density 5 × 10 ⁴ pfu / 150 mm plate and incubated at 47 ° C. for 3.5 h. Then
An IPTG (isopropyl-β-D-thiogalactopyranoside) -saturated nitrocellulose filter was placed on the plate and this was incubated at 37 ° C for an additional 3.5 h. After removing the filter and saturating the nonspecific binding site, it was incubated with undiluted B1 hybridoma supernatant for 1 h at RT. The second antibody was 1: 1 coupled to alkaline phosphatase (Dako).
000 diluted antibody-mouse antibody was used. Substrate reaction is B
Performed using CIP and NBT. After rescreening three times, positive plaques were cloned. After in vivo cleavage with helper phage R408, the pBluescript SK (−) plasmid containing the inserted B1 cDNA was isolated. Human kidney cDNA gene bank in phage MAX1 (Clonetech), density 3 × 10 ⁴ pf
U / 150 mm plates were plated and the plates were incubated at 37 ° C. for 4 h before placing nitrocellulose filters on the plates. After denaturing and neutralizing, the nitrocellulose filter was baked at 70 ° C. for 2 hours to fix the DNA. For both prehybridization and hybridization, 5 × Denhardt's solution, 0.5% SDS, 1M NaCl, 5 mM
EDTA (pH 8), 4 mM NaH ₂ PO ₄ , 37 mM
Performed at 42 ° C. in Na ₂ HPO ₄ , 10 mg / ml herring sperm DNA and 50% formaldehyde. pB1
the EcoR I / Hind III fragment of the cDNA
Radiolabeled with ³² Pa-dCTP by the "random primer" method and added to the hybridization solution.
After 16h, filter is 1x SSC + 0.1% SDS.
It was washed in the medium at 65 ° C. for 1 h and exposed to an X-ray film. From the 4 positive clones, 1 positive plaque remained after 3 rescreens. The inserted cDNA is Eco
This plasmid pYEU isolated after digestion with RI
ra3 is in vivo using helper phage R408
o Obtained from this plaque by cutting. This clone was named pB1.2.

B) Screening of genomic cosmid gene library The genomic gene library in cosmid vector pWE-15 contains human DNA from peripheral blood lymphocytes.
Elisabeth W, Professor of stitut fur Humangenetik in Munich
Granted by Dr. Eiss. NM5 with this cosmid DNA
44 bacteria were transformed by electroporation and 3 × 10 ⁴ colonies / 150 mm plates were transferred to nitrocellulose filters. After incubating at 37 ° C. for about 8 h, it was placed on the second nitrocellulose membrane to prepare a replica filter. The bacteria on the replica filter were denatured, then neutralized and the filter was baked for 2 h to immobilize the DNA and EcoR I as described for the λMAX1 gene library.
/ Hind III probe. One clone was isolated after three rounds of rescreening and its approximately 30 kb long insert was isolated after Not I digestion. This clone was named pWE-B1. BamH
After double digestion with I and BglII, a 5.9 kb fragment was isolated and ligated into the BamHI linearized pBSK (+) vector. This clone is pB1-C
It was named os.

C) DNA Sequencing All sequencing was performed on the double stranded plasmid DNA using the dideoxynucleotide method. 18-20 oligonucleotides having sequences derived from known nucleotide sequences were used in the sequencing reactions. Pharmaci
The T7 sequencing kit supplied by a ³⁵ S a-d
Adopted exactly according to the manufacturer's recommendations using CTP.
The reaction products were separated on a 6% polyacrylamide gel,
The gel was dried and then exposed to X-ray film.

D) DNA and RNA Blot Analysis RNA was isolated by the guanidine isothiocyanate method followed by ultracentrifugation in a cesium chloride gradient (Sambrook, J. et al.
Et al., 1989, Molecular Cloning: A Laboratory Manual, C
old Spring Harbor Laboratory, Cold Spring Harbor,
New York). For the purification of poly (A) ⁺ RNA, the PolyATtract system III kit from Promega using biotinylated oligo (dT) primer was used. The sample obtained by this method is 1.2% in 1 × MOPS buffer.
Fractionated on formamide / agarose gel, 20 × S
Blot on nylon membrane by capillary transfer method in SC buffer. 1% BS for both prehybridization and hybridization
A solution consisting of 1 mM EDTA, 250 mM Na ₂ HPO ₄ and 7% SDS (pH 7) is used. Blots were washed at the same temperature with a solution consisting of 1 mM EDTA, 20 mM Na ₂ HPO ₄ and 7% SDS (pH 7) and then exposed to X-ray film. DNA samples are obtained from peripheral blood lymphocytes previously purified by Ficoll gradients by taking cells in guanidine isothiocyanate buffer followed by ethanol precipitation. After digestion with the appropriate restriction enzymes, the DNA samples were fractionated with TAE buffer on a 0.8% agarose gel. Samples are depurinated in gels, denatured and then neutralized. Transfer and hybridization to nylon membranes is performed as described above for DNA blots.

F) Expression of MBP-B1 fusion protein The 798 bp pB1 cDNA insert was cloned into the Stu I cleavage site of the vector pMAL-c (Biolabs) in the appropriate reading frame. pMAL-B1
Transform TB1 bacteria (Biolabs) with the construct,
The resulting transformant had an OD ₆₀₀ of 0.5 at 37 ° C.
Incubated until To obtain the cell extract, 1 ml of the culture solution was taken, and the rest of the culture solution was 0.3 mM IPT.
Induction with G (final concentration). After incubation for 2 h, 0.5 ml of the induced bacteria were removed and two samples were treated with SDS loading buffer and fractionated in a 10% polyacrylamide gel (Sambrook et al., 1989).

G) Immunoblotting SDS PAGE-fractionated proteins were electroblotted (transfer buffer: 39 mM glycine, 38 m
M Tris, 0.037% SDS and 20% methanol), transferred to nitrocellulose, saturated the filters, and then with each antibody in TBS + 10% FCS (for tumor cell extracts) or TBS + 1% BSA (for bacterial lysates). Incubate. Alkaline phosphatase-conjugated immunoglobulin was always used as the second antibody and the substrate reaction was performed using BCIP and NBT tablets (Sigma).

H) Immunohistochemical Study 10 Using cryostat from human tissue frozen at low temperature
mm sections were prepared and incubated with undiluted B1 hybridoma supernatant. Anti-mouse, peroxidase-linked immunoglobulin was used as the second antibody. The substrate reaction was performed with H ₂ O ₂ in AEC buffer.

I) Isolation of MUC8 clones (of cDNA and genomic origin) Since biochemical purification of high molecular weight glycoproteins which allows N-terminal sequencing of proteins is often not possible, sequencing of cDNA clones and It is more practical to clarify the protein structure by feature analysis.

Monoclonal antibody B1 was used to screen the cloned cDNA gene library. After screening 2.4 × 10 ⁶ recombinant plaques, positive clones were isolated. This clone, designated pB1, contains a 798 bp long cDNA insert.

To obtain yet another nucleotide sequence, a radiolabeled pB1 cDNA fragment was used to screen a second human cDNA gene library prepared from kidney mRNA. Tested 6.3
After rescreening 3 times from × 10 ⁵ plaques,
The inserted DNA was named pB1.2, and the inserted DNA was 854 bp.
Positive clones containing

Since none of the cDNA clones were characterized by regions with repetitive sequences, but were not identical,
The genomic gene library was screened with the pB1 cDNA fragment. Of the 3 × 10 ⁵ colonies tested, one clone contained genomic DNA that hybridized to the cDNA. The insert of approximately 30 kb was cut with various restriction enzymes and hybridized with the two cDNA clones as probes in Southern blot analysis. The 5.9 kb long minimal fragment that could be isolated was subcloned into pBluescript and the resulting clone was designated pB1-Cos.

K) Sequence analysis of mucin clones Three clones, pB1, pB1.2 and pB1-C.
os All nucleotide sequences were determined by identifying sequence-specific primers for both DNA strands. p
The coding region of B1 cDNA is composed of about 10 repeat units of 53 bp each. Three of these repeats are found at the 3'end of the pB1.2 cDNA. All repeat units are characterized by an Ear I cleavage site. The repeat region of the B1 exon contained in the pB1-Cos clone is composed of 19 Ear I fragments. It has been heretofore possible to directly determine 225 bp by sequencing, as the repetitive sequence units do not give reliable results in the generation of sequence-specific primers or the assignment of sequences from Exo II deletion clones. Absent. The 321 bp at the 5'end of the pB1.2 clone and the 229 bp at the 3'end of the pB1 clone are not represented by sequences in the pB1-Cos exon. This indicates that at least two more exons containing these sequences must be present. This assumption is pB1-Cos
Exon is a classical A for intron / exon transposition
Confirmed by the fact that they are separated by the G-GT sequence. However, since the complete coding sequence for MUC8 mucin is contained within this one exon,
It can be used for cloning and expression of full length MUC8. The nucleotide sequence of that exon encodes 396 amino acids which, by computer analysis, provide a protein backbone of 4217 MW. The deduced amino acid sequence contains approximately 6 repeat units, each composed of 53 amino acids. Since the molecular weight confirmed by Western blot is about 170 kD, it is considered that oligomerization and / or strong co- and post-translational modification are performed as reported for other secreted mucins.

L) Blot analysis of genomic DNA Genomic DNA isolated from human donor peripheral blood lymphocytes
Samples were digested with EcoRI or Msp I and pB
Hybridize in a Southern blot with the EcoR I / Pst I fragment of one clone. All five samples showed distinct restriction fragment length polymorphisms, especially prominent in Msp I digestion. In this case the band varied between 1.5 and 2.9 kb in length. The size of the fragment from the EcoRI digestion was 10-12 kb. DNA samples containing human chromosome 7 from somatic cell hybrids also hybridized in the same size range.

M) RNA and Protein Blot Analysis 4 μg of poly (A) ⁺ RNA from various tumor cell lines
500 bp EcoRI / Hind III pB1 cDN
Hybridized with A probe in Northern blot. 3 of 4.8 kb, 1.9 kb and 1.36 kb
Two mRNA species were recognized, of which the 4.8 kb band showed the strongest hybridization. 5 '
Whether the banding pattern is due to a precursor or a degradation product of the 4.8 kb species cannot be determined at this time because the length of the untranslated portion and the transcription start point are unknown.

Under reducing conditions, a 170 kD-sized protein is recognized as B1 antigen on Western blots. Under non-reducing conditions, this band is about 40
It is 0 kD. The extent of its O-glycosylation is unknown. Consensus motif (Asn-X-Ser / Thr)
The possibility of N-glycosylation cannot be denied due to the absence of

[Brief description of drawings]

FIG. 1: Mouse anti-MUC8 Mab from human tumor cell line
Fig. 7 is a photograph of an immunoblot of a cell lysate isolated using B1. Proteins were electrophoretically fractionated on 3-10% SDS polyacrylamide gels by molecular weight and transferred to nitrocellulose. Undiluted hybridoma supernatant was used as the first antibody, and anti-mouse, alkaline phosphatase-conjugated immunoglobulin was used as the second antibody. Track 1: CAMA; Track 2: capan-1; Track 3: Mz-Sto-1; Track 4: MKN-4
5; Track 5: T84; Track 6: HT-29 M
TX.

FIG. 2. Nucleotide sequence of the pB1 cDNA clone, as well as the deduced amino acid (three letter abbreviation) sequence.
Sequences were determined using the Sanger method, generating sequence-specific primers using the T7 sequencing kit from Pharmacia.

FIG. 3: Bacterial culture medium (TB1) after transformation with pMAL-c vector or pMAL-B1
3 is a photograph of an immunoblot of a lysate isolated from
Bacteria were harvested before and after induction of the promoter by IPTG and pellets were taken up in SDS gel loading buffer.
After fractionation on a 10% SDS PAGE gel, the proteins were transferred to a nitrocellulose membrane and then operated as described in FIG. Track 1: IPTG induced pMAL-B1; Track 2: IPTG induced pMAL-c; Track 3: uninduced pMAL-B1; uninduced pMAL-c.

FIG. 4 is a homology comparison of amino acid sequences of two MBP-B1 fusion proteins, each of which is specifically recognized by Mab B1 and has a reading frame shifted by one nucleotide. The same amino acid motif was subjected to an analysis (Hopp & Woods, 1983) for calculating an antigenic determinant, and as a result, a QDREEE motif which may be an antigenic determinant was identified.

FIG. 5 is a photograph of Northern blot analysis using various human tumor cell lines. Purified poly (A) ⁺ RNA sample
It was fractionated by molecular weight in an agarose gel containing formaldehyde and transferred to a nylon membrane. Ec of pB1 clone
The oRI / HindIII fragment was used as a radioactive probe (A). β-actin probe (B) was used as a blot control. Track 1: HT
-29; Track 2: T84; Track 3: CAMA;
Track 4: Mz-Sto-1; and Track 5: C
apan-1.

FIG. 6 is a photograph of Southern blot analysis using human genomic DNA. Samples of genomic DNA (15 mg) from 5 different donors (1-5) were treated with EcoRI.
Completely digested with (a) or Msp I (b), 0.8%
Fractionation by molecular weight was performed on an agarose gel. The EcoR I / Pst I fragment of pB1 clone was used as a radioactive probe.

FIG. 7: Nucleotide sequence and deduced amino acid sequence of pB1.2 cDNA clone. The sequence was determined by the same method as described for pB1.

FIG. 8: Complete nucleotide sequence of genomic clone pB1-Cos compared to the two cDNA sequences pB1 and pB1.2. Exon / intron transpositions, and start and stop codons are highlighted in bold. The 225 bp in the unsequenced exons are underlined. Sequences were aligned using GeneWorks software from IntelliGenetics.

9 is a continuation of the nucleotide sequence of FIG.

FIG. 10 is a continuation of the nucleotide sequence of FIG.

11 is a continuation of the nucleotide sequence of FIG.

FIG. 12: pB1-Cos exon and two cDNAs.
Graphical representation of the A array. The same areas are arranged below each other. The display does not show the exact size.

FIG. 13: Partial digestion of the repeat region of pB1 PCR clone with the restriction enzyme Ear I. A: agarose gel,
B: EcoR I / Hind III as a radioactive probe
It is a photograph of an autoradiogram of a Southern blot using the pB1 fragment. Inserted DNA 2 mg in each case
Was digested under the following conditions. Track 1: 2 hours, 1U / m
g; Track 2: 5 minutes, 0.5 U / mg; Track 3: 3
Min, 0.5 U / mg; truck 4: 1 min, 0.5 U / mg;
M: 123 bp-standard length.

FIG. 14. Southern blot analysis with genomic DNA from human / rodent somatic cell hybrids. In each case, a 10 mg DNA sample was completely digested with EcoRI,
Fractionation on a 0.8% agarose gel. EcoR I / H
Shown are photographs of radiograms obtained using the indIII pB1 fragment as a radioactive probe. The samples are DNA containing human chromosome 1 in track 1 and DNA containing human chromosome 2 in track 2 (same below).
Were each loaded with DNA. Track 25: Human cell line IMR91; Track 26:
Mouse cell line 3T6; Track 27: Hamster cell line PJK88.

Figure 15: Western blot analysis with sera from pancreatic and colon cancer patients and healthy donors. Mz-Sto-1 cell extract was used as a positive control. 10 ml of each serum was loaded. 2 mg / ml Ma as the first antibody
b B1 was used as a second antibody with 1:50 anti-mouse immunoglobulin conjugated to alkaline phosphatase.
It is a photograph which shows the pattern of the Western blot used by diluting to 0.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location C12Q 1/68 7823-4B C12Q 1/68 A G01N 33/53 G01N 33/53 V 33/577 33 / 577 B // A61K 39/39 ADS A61K 39/39 ADS C12P 21/08 C12P 21/08

Claims (20)

[Claims] 1. A pCos nucleotide 3 of essentially FIG.
A cDNA encoding human mucin MUC8 corresponding to the nucleotide sequence of 049-4328. 2. A recombinant DNA containing the cDNA according to claim 1. 3. A mucin MUC8 prepared using the cDNA of claim 1. 4. A mucin MUC prepared using the amino acid sequence derived from the cDNA of claim 1.
8. 5. A cDNA according to claim 1 which essentially corresponds to the nucleotide sequence at nucleotides 3049-3325 of pCos of FIG. 6. The cDNA of claim 1 which essentially corresponds to the nucleotide sequence at nucleotides 3326-3666 of pCos of FIG. 7. The cDNA of claim 1 which essentially corresponds to the nucleotide sequence at nucleotides 3667-4238 of pCos of FIG. 8. A DNA essentially corresponding to the nucleic acid sequence pCos of FIG. 9. A DNA essentially corresponding to the nucleic acid sequence pB1.2 of FIG. 10. A DNA essentially corresponding to the nucleic acid sequence pB1 of FIG. 11. Use of the mucin according to claim 3 or 4 or an immunoreactive peptide derived therefrom.
A diagnostic method for detecting and quantifying a human antibody against mucin MUC8 as a tumor marker. 12. Use of the mucin or the immunoreactive peptide from mucin according to claim 3 or 4 in a method for detecting an antibody against mucin. 13. The method according to claim 11, wherein the detection is performed by ELISA. 14. Use of the mucin according to claim 3 or 4 or an immunoreactive peptide derived therefrom for inducing antibody formation in a mammal. 15. The use according to claim 14, wherein the mammal is a human. 16. A diagnostic method for detecting and quantifying mucin MUC8 as a tumor marker, using the sequence pCos in FIG. 8 or a constituent sequence derived therefrom as a gene probe. 17. Use of the DNA according to one of claims 1 and 2 and 5-10 for the recombinant production of mucin MUC8 or its constituent sequences. 18. A mucin MUC8 or a peptide derived therefrom, which can be synthetically produced based on the amino acid sequence derivable from the cDNA of claim 1. 19. A monoclonal or polyclonal antibody against mucin MUC8 obtained after immunization with MUC8 according to claim 3 or 4. 20. A diagnostic reagent for detecting MUC8, which comprises one or more antibodies against MUC8 according to claim 19.